1. Ethics
This study was approved by Ethics Committee of Zhengzhou Central Hospital Affiliated to Zhengzhou University (ethics number: 202126).All experimental procedures in rabbits were conformed with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH Publication No. 85-23, revised 1996). Sexually mature female New Zealand Rabbit (2800-3000 g) at the age of 14-16 weeks were purchased from Pizhou Dongfang breeding Co., Ltd(permit number: SCXK(su)2017-0002), Jiangsu, China. All rabbits were fed rabbit chow and water ad libitum under temperature controlled environment at 25℃ with a 12 h light and dark cycle for 1 week.
2. Isolation of hUCB-MSCs
Umbilical cord blood samples (about 50mL each) with anticoagulant (EDTA) were collected from umbilical cord vein. Mononuclear cells (MNCs) were isolated by density gradient centrifugation for 30 minutes at 400 g and washed 3 times in PBS (Beyotime). The isolated MNCs were plated in 75cm2 cell culture flask containing mesenchymal stem cell basal medium (Yocon) and incubated at 37°C in a humidified atmosphere of 5% CO2. Non‑adhering cells were removed after adherence phenomenon, and then medium was changed every 3 days. After expansion to 80~90% confluence, the cells were harvested by 0.25% trypsinization and subcultured for further experiments. Inverted microscope was used to analyze the morphology of hUCB-MSCs. According to the International Society for Cellular Therapy, MSCs have three characteristics: (1)plastic-adherent, (2)express CD105, CD73, and CD90 and not express CD45, CD34, CD14, CD11b, CD79α, CD19, and HLA-DR surface antigen, (3)differentiate into osteoblasts, adipocytes in vitro [21]. Thus hUCB-MSCs were analyzed by flow cytometry, osteogenic and adipogenic differentiation assays.
3. Flow Cytometric Analysis
Detached hUCB-MSCs were washed twice with ice-cold PBS, centrifuged, and fixed in 4% paraformaldehyde. Then cells were incubated with mouse anti-human CD45-FITC, CD34-FITC, CD11b-FITC, CD19-FITC, CD29-FITC, CD73-PE, CD105-APC-A750, CD90-APC and HLA-DR-FITC (all from BioLegend) in dark for 20 minutes. Cells were analyzed by flow cytometer (Beckman Coulter GmbH, Krefeld, Germany).
4. Osteogenic and adipogenic differentiation
To investigate the osteogenic and adipogenic differentiation potential of hUCB-MSCs, third-passage cells were plated at a concentration of 3´103cells/cm2and cultured with osteogenic and adipogenic medium for 3 weeks with medium changes twice weekly, respectively. Osteogenic medium consists of IMDM supplemented with 0.1mM dexamethasone, 10 mM β-glycerolphosphate (Sigma-Aldrich), and 0.2 mM ascorbic acid. Adipogenic medium consists of IMDM supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 1mM hydrocortisone (Sigma-Aldrich), 0.1 mM in domethacin(Sigma-Aldrich), and 10% rabbit serum (Sigma-Aldrich). At the end of differentiation, cells were stained with Alizarin Red S and Oil Red O respectively.
5. The establishment of IUA model
Rabbit IUA models were constructed according to Liu et al [22]. Rabbits (n =9) were randomly subdivided into 3 groups, including control group (n = 3, rabbits without any treatment), 14 days after surgery group (n = 3, rabbits underwent IUA modeling surgery, sacrificed on 14th day), 28 days after surgery group (n = 3, rabbits underwent IUA modeling surgery, sacrificed on 28th day).
All rabbits in model group were anesthetized with urethane (1.5g/kg) through ear venous. They were then placed in a supine position and the lower abdomen was shaved and sterilized with 70% ethanol on the operating table. A vertical incision (2.5-3 cm) was performed and the bilateral uterine horns were exposed (Fig.1E). A 0.5-cm longitudinal incision was scissored in the uterus to scrape the inner endometrium with a small curette until feeling rough. After curettage, a LPS surgical suture placed in the uterine cavity (Fig.1F).The uterine cavity and peritoneal cavity were thoroughly rinsed with physiological saline, then the abdomen was sutured (Fig.1G). The LPS surgical suture was removed at 48 hours after surgery. On 14thand 28thdayafter surgery, 3 rabbits were sacrificed for the collection of uterine tissue in model group, respectively.
6. Treatment for IUA model
The rabbits (n = 6) were randomly assigned to two groups, including estrogen treatment group (n = 3, rabbits underwent surgery of intrauterine adhesions, estradiol benzoate (0.5mg/kg) was administered intramuscularly every 4 days) and hUCB-MSCs treatment group (n = 3, rabbits underwent surgery of intrauterine adhesions, 1 weeks after surgery, a relaparotomy was performed and the rabbits were injected with 1´106 hUCB-MSCs in each of the uterine).After 28days of treatments, all rabbits were sacrificed for the collection of uterine tissue.
7. Histological analysis
The endometrial gland number and fibrosis rate were examined via hematoxylin-eosin (HE) and Masson staining respectively. The excised uteri were fixed in 4% paraformaldehyde, then embedded in paraffin, sliced into 5-mm thick sections, and routinely stained with HE and Masson stains according to standard protocols. Sections were examined under an inverted microscope (Leica, German, DMIL-PH1). Four high-power fields (HPF) were selected on each HE-stained slice to count the number of glands. Four high-power fields (HPF) were selected on each Masson-stained slice to calculate the degree of endometrial fibrosisusing Image J software.
8. Immunohistochemistry
In order to evaluate expressions of endometrial receptivity related estrogen receptor (ER), angiogenesis related Ki-67, and inflammation related transforming growth factor-β1 (TGF-β1), immunohistochemistry was used. The transverse paraffined uterine sections were deparaffinized, rehydrated, and then incubated in 5% bovine serum albumin (Beyotime) for 30 min at 37℃ to block the nonspecific antibody. Sections of the uterus were deparaffinized and gradually dehydrated. Slides were incubated with rabbit anti-Ki-67 (1:200 dilution),anti-ER (1:200 dilution), and anti-TGF-β1 (1:200 dilution) monoclonal antibody at 4℃ overnight. And then the sections were incubated with goat anti-rabbit secondary antibodies for 60 min at room temperature and the reaction was stopped with 3,3-diaminobenzidine. Image J software (National Institutes of Health) was used to evaluate the expression of Ki-67, ER, and TGF-β1-positive cells, respectively, at a magnification of 400´. Five fields were randomly selected from each slide to determine the mean optical density (MOD).
9. Single-cell RNA sequencing (scRNA-seq)
A rabbit IUA sample with hUCB-MSCs transplantation was utilized to single-cell RNA sequencing and bioinformatics analysis. The dually edited Mel-RM (Mel-RM.DE) cells were serum starved for 96 h and the endometrial quiescent cells were sorted, washed twice and re-suspended in cold PBS (calcium and magnesium free) with 0.04% FBS. Cell number and viability were determined using hemocytometer and Trypan Blue staining and 1´105 cells were subjected to10´ Genomics sequencing according to the manufacturer’s protocol by Shanghai OE Biotech co., LTD (Shanghai, China). Briefly, viable endometrial cells isolated from dually edited Mel-RM cells after serum starvation were analyzed using the 10´ Genomics Chromium Droplet platform with unique transcript counting through barcoding with unique molecular identifiers (UMIs). Cell Ranger 3.1.0 and Seurat 3.1.1 were used to analyze the sequencing results.
Considering that the rabbit IUA sample was treated with hUCB-MSCs, we mapped the sequenced reads to the human and rabbit reference genomes respectively, obtaining 4363 human derived cells and 10,599 rabbit derived cells. After quality filtering to remove cells expressing high mitochondrial gene signatures and excluding doublets, 4097human derived cells and 8792 rabbit derived cells were retained for further analysis. Upon gene expression normalization for read depth, cells were subjected to t-distributed stochastic neighbor embedding (t-SNE) and several unsupervised cell clusters were obtained and visualized using Loupe Browser. The cluster-specific markers were identified by detecting the differentially expressed genes between the given cluster and the other clusters.
Pseudotime trajectories were constructed with MONOCLE (version 2.6.4) (Qiu et al., 2017). Briefly, we first selected a set of ordering genes which showed differential expression between clusters. Then, Monocle uses reversed graph embedding, a machine learning technique to learn a parsimonious principal graph, reduces the given high-dimensional expression profiles to a low-dimensional space. Single cells are projected onto this space and ordered into a trajectory with branch points. As called in Monocle, cells in the samesegment of the trajectory have the same ‘state’.
10. RNA Expression Profiling by RNA Sequencing
The rabbit IUA samples with and without treated with hUCB-MSCs were used for transcriptome sequencing (3 vs 3). Firstly, total RNAs were extracted by Trizol method, RNA purity was detected by spectrophotometer, RNA integrity was analyzed by agarose gel electrophoresis and Agilent 2100 BioAnalyzer. The Library was constructed using Illumina’s NEBNext® UltraTM RNA Library Prep Kit. Then, Illumina platform was used for library sequencing and 150 bp paired terminal reading was generated to obtain the sequence information of the fragment to be measured. After quality control and sequence alignment based on reference genome, DESeq2 software [23] was used to analyze the differentially expressed genes (DEGs) between the two groups. Finally, the DEGs was used for gene enrichment analysis based on gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG).
11. Western Blot
Proteins were extracted from endometrium samples with RIPA buffer containing proteinase inhibitors. The protein concentrations were quantified using BCA Protein Assay Kit (Beyotime, Shanghai, China) and separated in SDS-PAGE gel (Solarbio, Beijing), then they were transferred onto the PVDF membrane. The membranes were incubated with anti-NF-kB-p65 and anti-GADPH for overnight at 4℃, followed by secondary antibodies for 1 h at room temperature. The blots were visualized using ECL chemiluminescence kit (enhanced), and the band intensity was quantified with Image J software.
12. Statistical Analysis
Statistical analysis was performed with SPSS 20.0 software. Numerical data were indicated as means standard deviation. For nonparametric statistics, data were analyzed using the Mann Whitney U test and presented as populations with median values indicated by bars. For parametric statistics, data were analyzed using unpaired Student Ttest. Data was presented as a mean value with 95% confidence interval (CI). P values <0.05 were considered to be statistically significant.